Breeding commercially adoptable cotton resistant to reniform nematode

Introduction

Soil-borne parasitic nematodes are a major biotic constraint for global cotton production, because they can significantly reduce cotton productivity via acting alone and/or interacting with soil-borne pathogens, for example, Fusarium, Verticillium, Rhizoctonia solani and Berkeleyomyces basicola (Koenning et al. 2004). Renifom nematode (Rotylenchulus reniformis), as one of four nematode species pathogenic to cotton, is reported to be the most destructive and has increased its spread and impact (Koenning et al. 1996, 2004; Robinson 2007). In the recent two decades, for example, the reported surveys have suggested that reniform has gradually replaced its root-knot counterpart (Meloidogyne incognita) in Mississippi, Louisiana and Alabama in the USA, where the latter was historically the major concern (Singh et al. 2023). Reniform can infest the roots of cotton as early as at seed germination, resulting in stunting and dead plants and subsequently a 40–60% reduced crop yield (Robinson 2007).

The reniform nematode is a sedentary semi-endoparasite but can infect and live with a broad range of plant species, ranging from most field crops to vegetables and weeds species (Koenning et al. 2004; Khan 2005; Robinson 2007). The juveniles from hatching to the J4 stage remain inside the cuticles of the moults to grow into vermiform immature adults. After emerging free, only the females penetrate the cotton root cortex and establish a permanent feeding position in the stele of the roots to steal and compromise plant nutrients, water and carbohydrates and their transportation (Koenning et al. 2004; Robinson 2007). Reniform is highly adaptive against cold, hot and drought conditions and can live in different soil types to a depth of 1 m or more. Its ability to live and survive in deep subsoils increases the chance of its rapid return in topsoils, even under the practice of treating the topsoils with nematicides at planting (Robinson 2007). Similarly, crop rotation systems, either with poor and non-host crops, such as corn, sorghum, wheat, barley and sunnhemp or fallow, can reduce nematode population in soils but the effect was reported to be varied and/or short-lived, as the population often rebounded quickly when the fields returned to plant cotton (Davis et al. 2003; Koenning et al. 2004; Khan 2005; Starr et al. 2007; Smith et al. 2024). These abilities together with its short life cycle make it a most challenging pest to manage and control.

The search and deployment of genetic solutions have been pursued to overcome the above challenges and to find cost-saving and durable control measures for reniform nematode in cotton. The efforts led by USDA researchers concluded that moderate, but largely insufficient, resistance was present within Gossypium hirsutum (Weaver et al. 2007; Gaudin and Wubben 2021); however, there was high resistance to immunity in diploid cotton species, particularly the A, D, E and F subgenomes as well as undomesticated G. barbadense (AD2 genome) (Robinson 2007; Starr et al. 2007; Khanal et al. 2018a). Interspecific breeding was conducted to transfer different sources of resistance into G. hirsutum backgrounds and today a number of resistances are available and can be exploited in developing resistant cotton varieties (Khanal et al. 2018a; Singh et al. 2023). Of them, Ren^lon and Ren^barb are the two most popular being studied and used extensively. The former originates from G. longicalyx (F) and was incorporated into G. hirsutum first by using a bridge-crossing approach to develop a triple-species hybrid followed by many generations of backcrossing (Robinson et al. 2007; Bell et al. 2014). The resistance is controlled by a single dominant gene and confers plant immunity from reniform infestation. However, it induces a plant intolerant response, particularly when used in heavily infested conditions and causes self-harm to plant root cells, which makes the plants themselves less able to take up water and nutrients, hence plants become stunted (Sikkens et al. 2011). Therefore, the use of Ren^lon becomes questionable for commercial exploitation; however, research is continuing to use it in combination with other control measures, for example, nematicide treatment (Schrimsher et al. 2014), and also even with other resistance genes, i.e. Ren^barb (Wubben et al. 2017; Gaudin et al. 2020). Ren^barb originates from an undomesticated G. barbadense accession, GB713 (Gutiérrez et al. 2011), and is controlled by two major quantitative trait loci (QTLs), namely, Ren^barb2 and Ren^barb3, with the former being a major contributor to suppress egg production (Gutiérrez et al. 2011; Wubben et al. 2017; Gaudin et al. 2020). In infested fields, Ren^barb showed high resistance to the parasite without intolerant responses to hamper plant growth and development (Sikkens et al. 2011; Schrimsher et al. 2014). Ren^lon and Ren^barb were mapped in different chromosomes in cotton and simple-sequence-repeat (SSR) markers developed (Dighe et al. 2009; Gutiérrez et al. 2011) and stacking them together did not offer any additive resistance (Gaudin et al. 2020). Cotton breeders in USA currently use Ren^barb in developing and releasing new resistant cotton varieties, but agronomic performance of these resistant lines requires further improvement (Singh et al. 2021).

The backcross breeding method is most commonly used to transfer novel traits of interest in exotic germplasm into elite locally adapted varieties in plant breeding (Briggs and Allard 1953). The traits are usually simply inherited and/or highly heritable when they belong to quantitative traits. In cotton breeding, the method was used to incorporate new disease resistance genes from non-adapted to adapted germplasm (Bayles et al. 2005), to improve fibre strength (Meredith 1977), and in this transgenic cotton era, to incorporate and stack genetically modified (GM) traits into elite cotton varieties (Zhang 2015). The method was used successfully to transfer nematode resistance from other species into G. hirsutum (Dighe et al. 2009; McCarty et al. 2013, 2017; Bell et al. 2014, 2015, 2023) and to develop new varieties (Singh et al. 2023). The goal of the approach is to develop new varieties competitive to the elite recurrent parent (RP), but with the add-on of resistance from a donor parent; thus, the new version is expected to outperform susceptible commercial varieties grown in the nematode-infested farms while remaining competitive when grown in non-infested farms. Generations of backcrossing and alleles in the donor parent closely linked to the trait of interest can have an impact on the outcome of the approach (Meredith 1977; Bayles et al. 2005) and further understanding is also required on how these factors can affect the outcome in breeding cotton resistant to reniform nematode.

Two species of reniform nematode, namely, R. reniformis and R. parvus, exist in Australia (Colbran 1960). Only R. reniformis causes damage to crops, such as pineapples, vegetables, and sweet potatoes in northern Queensland. It was first detected in a cotton field in 2003, near Emerald in central Queensland, followed by confirmation of its spread and impact in cotton in the Dawson Valley since 2012 (Smith et al. 2024). After importing two resistance sources from USDA, the CSIRO cotton breeding program evaluated their effectiveness and relevance to local reniform species, meanwhile looking for the best strategy of executing breeding resistance to local reniform nematode in cotton. After a comprehensive review on the current impact state of the parasite, the remoteness of infested region to key cotton production regions and also the need of strict adherence of industry biosecurity policy, it became apparent that our breeding effort for the resistance cannot rely on a phenotyping bioassay under the controlled environment to identify resistance and susceptibility in segregating generations of breeding populations, as the other programs are doing (e.g. Robinson et al. 2007). In contrast, given the availability of molecular markers for introducing resistance, we chose an alternative approach to develop resistant cotton, which comprises first using marker-assisted selection to track, select and develop homozygous resistant lines, followed by the agronomic evaluation of the new lines in non-infested field environments to select for improved yield and fibre quality. We demonstrated that such an approach could develop new resistant varieties that are competitive in agronomic performance against the commercial varieties. Thus, when released, they would provide an effective control measure for reniform nematode infested farms and for the industry, so as to restrict and minimise the impact and spread of the parasite while not compromising farm productivity and economic return. In this study, we share the results of that breeding practice.

Materials and methods

Breeding scheme for cotton resistance to reniform nematodes

We integrated backcrossing with marker-assisted selection to transfer reniform nematode resistance from two introduced sources into our elite genotypic backgrounds. Fig. 1 shows an example scheme we used to transfer Ren^lon into an elite conventional line. LONREN-2 was a donor with a normal leaf type, and it had a good performance in our initial field test; 67102OL-117 with okra leaf was used as the recurrent parent (RP). Four times of backcrossing (BC) were conducted throughout. Resistant plants were screened and selected in a segregated family of BC₂F₁, BC₃F₁ and BC₄F₁ generation, and advanced via selfing in the successive generations until they became homozygous. At the end, all derived lines from different BC generations were tested together in field experiments for two consecutive years from 2019/2020.

Fig. 1.

Breeding scheme and timeline for transferring reniform nematode resistance from a donor parent, LONREN-2 (Ren^lon), into a current parent, 67102OL-117, via backcrossing. Resistant plants were tracked and selected in each of three backcrossing generations, i.e. BC₂F₁, BC₃F₁ and BC₄F₁, and from them, resistant lines were derived, confirmed and advanced by selfing to become homozygous. All these lines were finally tested together in field experiments in 2019/2020 and 2020/2012. ⊗indicatess selfing.

In the 2016/2017 summary, a conventional BC₂F₃-derived resistant line with Ren^lon was selected as a donor to cross with a transgenic breeding line containing the B3XF traits, to develop transgenic lines with nematode resistance. The resultant F₁ hybrid was further crossed with another F₁ hybrid from our B3XF breeding pipeline, to add on and enforce more genetics of our elite germplasm (Supplementary Fig. S1). The final cross was advanced through selfing up to F₄ and screening was conducted in a segregating population in a similar manner as described above to select plants with homozygote for transgenic traits as well as Ren^lon. The seeds from resistant plants were finally harvested and bulked together, and they were space planted and grown as a population in a non-infested field. After confirming resistance status, single plants were harvested and selected on the basis of lint percentage and fibre property measurements of the high-volume instrumentation system (HVI). The derived lines from the retained individual plants were tested, culled and progressed according to the process described in backcross scheme (Fig. 1) in every successive season, poor performers were discarded based on yield and fibre properties in the field test and the retained lines were re-tested in the follow-up season. At the end, the six best candidate lines were tested in the replicate trials over 12 locations in the 2020/2021 and 2021/2022 season. Two of them were kept and used in an infested field experiment above to validate the effectiveness of the resistance.

A backcrossing scheme similar to that for Ren^lon was used to transfer Ren^barb from M713 Ren 5 into an elite breeding line containing the B3XF traits. However, the process was complicated with the number of independent traits to be stacked together, when both two major QTLs for Ren^barb2 and Ren^bar3 were equally targeted. To reduce the complication and save time, a decision was made to focus only on Ren^barb2 (referred as Ren^barb through the paper for simplicity), while leaving Ren^barb3 unscreened, given the latter only attributed minor resistance (Wubben et al. 2017). Three generations of backcrossing were conducted and selection for plants with the presence of nematode resistance and GM traits was conducted in the BC₂F₁ and BC₃F₁ generations and they were advanced through selfing in glasshouse, until all traits were homozygous. Following a similar process to that used to develop transgenic lines with Ren^lon aforementioned, bulked seeds from single homozygous plants were space planted and screened for the confirmation of resistance in a non-infested field. The derived lines were selected and tested seasonally, and, finally, those best performers were tested through two seasons over multiple test sites across cotton production regions free from nematode infestation.

Results

Effectiveness of introduced resistance

At pre-planting, the initial reniform population was lower in the number in the field used in 2016/2017 than in 2021/2022 (Fig. 2). All four introduced resistant lines were effective in suppressing the increase of reniform population during the early crop-growing period up to mid-season when the experiment was terminated. There was a substantial increase (≥1000-fold) of reniform nematode in the soil samples planted with two susceptible commercial varieties (Fig. 2a). In comparison, LONREN-2 was less effective than LONREN-1, and also M713 Ren 1 and 5, in containing the resistance from G. barbadense.

Fig. 2.

Nematode population in soil samples prior to planting and at plant cut-out stage in field plots planted with (a) the introduced, or (b) locally bred resistant lines and susceptible varieties. Two LONREN lines carry a Ren^lon resistance; two M713 lines have both Ren^barb2 and Ren^barb3 resistance. Breeding lines, CSX3111 and 3279, have Ren^lon resistance; the lines, CSX3206, 3291, 1284 and 1347 have Ren^barb2 from M713 Ren 5, but whether they also have Ren^barb3 was unclear, because that QTL was not screened, tracked and selected; Sicot 71 and Sicot 746B3F in the upper panel (a) and Sicot 761B3XF and Siokra 253B3XF in the low panel (b) are susceptible commercial varieties. Different letters between the means of prior-sown and cut-out stage for each test lines suggest their significant difference at P = 0.05; error bars represent the standard error of the mean. B3F, Bollgard 3 Roundup Ready Flex®; and B3XF, Bollgard 3 XtendFLEX®.

Newly bred resistant lines were similarly effective in suppressing reniform population, i.e. a significantly reduced gain of reniform population in plots grown with resistant cotton over the crop period to mid-season, in contrast to a substantial increase in plots grown with susceptible controls (Fig. 2b). This is particularly evident in four lines with Ren^barb from M713 Ren 5, because the parasite population remained unchanged throughout two soil sampling times. In two lines with Ren^lon from LONREN-2; however, the increase was still statistically significant, but not to the same extent as what was observed in the soil samples where two susceptible controls were grown.

Plant measurements taken after 3 months of planting suggested taller plants in the most resistant lines with Ren^barb (Table S4). This was also accompanied with longer internodes and higher accumulated fresh biomass/m². In contrast, two lines with Ren^lon and Sicot 761B3XF had shorter plant and/or lower fresh biomass; therefore, they exhibited less early growth.

Effect of backcross generations on agronomic performance

BC families with Ren^lon resistance

Lint yield gradually shifted higher in response to the number of times of backcrossing, whereas the variability remained comparable among the families derived from different BC generations (Fig. 3). Mean yields of BC₃ and BC₄ families were close to the RP, and they also contained individual lines that outyielded the RP. When yield gain across different backcross generations was converted as the recovery rate of the RP, it showed a strong relation with the theoretical (Fig. S2). The recovery rates matched each other in BC₃, and were better than the theoretical in BC₄, but lower than the theoretical in BC₂, which may suggest some challenges achieving the expected recovery in early backcrossing generations.

Fig. 3.

Mean and variation of lint yield in three backcrossing generation-derived families with Ren^lon resistance when tested in non-infested fields. Blue and black dashed lines represent lint yield of a recurrent parent, 67102OL-117, and a donor, LONREN-2. The value below each boxplot represents the number of test lines.

The effect of successive backcross generations on fibre properties was not as apparent as seen in lint yield (Fig. 4). Mean fibre length never achieved the level of the recurrent parent. For both fibre strength and micronaire, the RP and donor had a small difference, and there was also no apparent gain or loss with the times of repeated backcrossing; however, all BC families had higher means than their two parents. This phenomenon was more obvious for fibre strength and micronaire, when four best-yielding lines from different BC generations were compared (Table S5). Therefore, the resistant donor contributed favourable alleles to improve these fibre properties, which agreed with the previous finding that LONREN lines had better fibre strength and finer fibre (Bell et al. 2014). Our evidence also indicates that the positive contribution from the donor did not diminish with the times of repeat backcrossing, which drove a proportional reduction of donor genetics in the resultant families and lines.

Fig. 4.

Mean and variation of three fibre quality properties in the families from different backcrossing generations with Ren^lon resistance when tested in non-infested fields. Blue and black dashed lines represent mean fibre quality properties of a recurrent parent, 67102OL-117, and a donor, LONREN-2.

Not all transgenic lines with the resistance were derived from backcrossing, but from a cross of two hybrids sharing a common elite parent (Fig. S1). When the best lines were tested in non-infested farm sites, they exhibited 6–9% lower lint yield than the control, Sicot 761B3XF, but possessed improved fibre properties (Table S6). One explanation is linkage drag and/or pleiotropy may still act with Ren^lon (Robinson et al. 2007). Two of these lines also showed less early growth vigour when grown in an infested field (Table S4), which partially supports the previous finding of the association between stunted plants and the presence of Ren^lon (e.g. Sikkens et al. 2011). All the above findings made us conclude that continued work with Ren^lon was not viable; so, no further work was conducted.

BC families with Ren^barb resistance

A similar number of derived lines from BC₂ and BC₃ were initially tested in a non-replicated progeny row experiment, after they were derived from retained individual plants. Subsequent selection resulted in a greater number of lines retained from the BC₃-derived (42) than BC₂-derived (15) family (Table S3); one more backcrossing, therefore, was able to improve both yield and fibre properties. When selections were tested in multiple sites over two seasons, the superiority of BC₃- over BC₂-derived families remained unchanged, and, more interestingly, within the BC₃-derived family, there were lines that yielded as high as the best control, Sicot 619B3XF (Table 1, Fig. 5).

Table 1.Yield and fibre properties of best transgenic lines selected from two backcrossing generation-derived families with Ren^barb resistance, compared with two susceptible commercial varieties based on seven non-infested site testing over two seasons.

Line	Backcross generation	Lint yield (kg/ha)	Lint percentage (%)	Length (mm)	Strength (g/tex)	Micronaire
CSX3215	BC2	2962a	42.6bc	32.1ab	31.5b	4.4a
CSX3321	BC2	2963a	41.8cd	32.4a	33.2a	4.2bc
CSX3206	BC2	2583b	40.8d	32.4ab	33.2ab	3.6f
CSX3291	BC2	2516b	41.0d	32.7ab	32.6bc	3.5f
CSX1126	BC3	3061a	41.3d	32.1ab	33.6a	4.1c
CSX1228	BC3	3068a	43.4b	32.0ab	33.8a	4.2bc
CSX1443	BC3	3045a	42.2cd	32.3a	31.6b	4.1c
CSX1484	BC3	3101a	43.1bc	31.8ab	33.5a	3.9d
CSX1347	BC3	2980a	42.4bc	33.3a	34.4a	3.8d
CSX1284	BC3	2807a	42.6bc	32.3ab	32.9b	3.8d
Sicot 619B3XF		3018a	42.3cd	29.9b	32.9ab	4.1c
Sicot 761B3XF		3010a	44.5a	30.9b	32.5ab	4.3ab
l.s.d.0.05		221.7	1.02	1.30	1.39	0.20

B3XF, Bollgard 3 XtendFLEX®.

Different letters in the same column indicate a significant difference at P = 0.05.

Fig. 5.

Mean and variation of lint yield in the families derived from two backcrossing generations with Ren^barb resistance when tested in non-infestation fields over different regions. Blue dashed line represents lint yield of the best control, Sicot 619B3XF, in the experiments. The values below each boxplot represent the number of test lines. B3XF, Bollgard 3 XtendFLEX®.

Yield and fibre properties from non-infested field test of best yielders and those tested in an infested field in 2022/2023 are given in Table 1. Except for two lines from BC₂ family, other lines had comparable yield one to another and to the controls, with means of those BC₃-derived lines being higher in general. Lint percentage was comparable to the control, Sicot 619B3XF, but lower than that for Sicot 761B3XF. Fibre quality was overall better than the controls.

Discussion

The invasion and establishment of reniform nematode in the northern traditional cotton production regions in Australia has signalled its potential to become a major threat to the entire industry (Stirling 2023; Smith et al. 2024). Concurrently, there is also renewed interest in developing cotton production in northern Australia, where reniform nematode has been known to exist for a long time (Stirling 2023). It is likely that both these developments will inevitably lead to accelerated parasite spill-over and spread into other non-infested regions, under today’s farm practice and logistics (Robinson 2007). Therefore, there is urgency to develop and implement a comprehensive management strategy for reniform nematode in Australia. The strategy should be composed of all aspects from farm gate hygiene, crop rotation, weed control, nematicide application to use of host plant resistance (Robinson 2007; Starr et al. 2007; Singh et al. 2023; Smith et al. 2024). In this study, we first attempted in Australia to understand the effectiveness of two popular resistance sources from overseas to local parasite and to undertake initial breeding efforts to develop high-performing reniform nematode resistant cotton.

Our results supported that both sources of resistance, either in their overseas or domestic elite genotypic backgrounds, were effective in suppressing local reniform reproduction when grown in infested field conditions. Meanwhile, Ren^lon from LONREN-2 was not as effective as Ren^barb, which is inconsistent with previous studies in the USA (e. g. Bell et al. 2014; Gaudin et al. 2020). The observation also contradicts higher effectiveness of LONREN-1 in the same experiment. This inconsistency agrees with the findings of the study of Schrimsher et al. (2014), but disagrees with the evidence that both LONREN lines share the same breeding origin and their resistance is reported to confer plants immune to reniform (Yik and Birchfield 1984; Robinson et al. 2007; Bell et al. 2014). One explanation for that may be due to the interaction of the Ren^lon resistance with local reniform nematode population. The variability of reniform nematode was reported in the populations collected in different regions of the USA and other countries and the same was the case for the existence of different pathogenicity (Agudelo et al. 2005; Khanal et al. 2018b). Therefore, further studies are needed to understand the interactions of local reniform population with different resistance genes.

It is noteworthy that for the resistance carried in M713 Ren 5 background, this study focused only on tracking and selecting Ren^barb2, owing to its major role behind the resistance (Wubben et al. 2017) as well as for reducing the number of traits targeted in backcrossing introgression process. It is interesting that our results confirmed that all derived resistant lines were highly effective in reducing the build-up of reniform population in an infested field. Wubben et al. (2017) demonstrated that the isogenic line with both Ren^barb2 and Ren^barb3 QTLs offered increased suppression on reniform egg production. Therefore, the questions remain on whether both resistant regions should be pyramided for better control on local reniform nematode and how that may affect agronomic performance of derived lines.

In this study, the backcross breeding method was used to transfer the resistance from two imported sources into our elite germplasm backgrounds. We relied on KASP marker-assisted selection to track and screen the resistance and conducted repeated backcrossing of selected resistant plants to reconstitute the genetics of the RP. This approach allowed selection of lines in non-infested conditions without the need of lab and field phenotyping for resistance, which subsequently saved time and resources. On the basis of the confirmation test of infested field experiments, KASP markers re-developed from the initial SSR markers or corresponding genomic regions for the resistance (Dighe et al. 2009; Gutiérrez et al. 2011; Bell et al. 2014) are reliable. Other researchers used an approach similar to that used for developing resistant germplasm in this study (Gutiérrez et al. 2011; McCarty et al. 2013). More interestingly, for Ren^barb, the markers we used for selection covered much wider genomic region than that flanked with SSR markers (Gutiérrez et al. 2011). That may imply that resultant lines will contain a relatively large genetics of G. barbadense after introgression of the resistance. Deleterious genes in that introgressed region will no doubt affect agronomic performance of the resultant lines, if they exist. A good example is the phenomenon observed for Ren^lon in this study (Table S6). However, that does not appear to be the case for Ren^barb, because relying on field evaluation, there was still a good chance of identifying outstanding lines with yield and fibre quality competitive to the commercial control varieties (Table 1). Nevertheless, further research is needed to confirm whether genomic region hosting Ren^barb2 is benign to agronomic performance or not. Now such work should become easier to do with the recent progress made in identifying its likely candidate genes (Wubben et al. 2025).

The goal of our breeding effort is to develop reniform nematode-resistant cotton with agronomic performance similar to that of the current commercial varieties. Our approach also allowed breeding and selection focusing on agronomic performance within the BC-derived families, once the resistance was incorporated. This increased the likelihood of selecting outstanding lines as evident in our results (Tables 1, S5). However, with this approach, there were challenges. The most important one is undesired agronomic traits of the donor parents associated with the resistance. In this study, this is particularly evident for Ren^lon resistance. Repeated backcrossing has been suggested to facilitate the recombination of genomic regions associated with resistance, because that undesired linkage with the traits will be broken, and to increase performance of the resultant progenies. However, for Ren^lon resistance, this did not appear to occur; even resistant germplasm containing more local genetic background was used in transgenic breeding pipeline to develop better-performing transgenic resistant cotton. If this is due to linkage drag and/or pleiotropy, reducing the introgressed chromosome genomic segment should be attempted. Alternatively, breeders should evaluate alternate sources of resistance. In this study, we did not see any significant challenges imposed by Ren^barb on agronomic performance, despite the fact that a relatively large coverage of its flanking KASP markers may potentially bring about unexpected genetics from G. barbadense affecting agronomic performance as discussed aforementioned. The other important matter for breeding is the recovery of the RP after each repeated backcross. In this study, we observed a lower-than-expected recovery for lint yield in the families from the second backcrossing with Ren^lon resistance, despite this was not occurring in the other BC generations. This is likely to be due to insufficient or unrepresentative progeny plants being selected for successive generation backcrossing. It is known that there is a larger segregation in early generations of backcrossing. In our current breeding practice, we still rely on phenotypic selection and empirical experience to decide the number of plants being selected for successive backcrossing, and the occurrence of the above phenomenon is expected. Frisch and Melchinger (2005) used simulation to demonstrate this risk. Therefore, a better alternative is to use a marker-assisted backcross breeding strategy guided with genomic prediction and simulation, because this will assist plant breeders to assess, identify and select individual progenies with higher genetic worth for successive BC, intermating or both (Visscher et al. 1996; Ni et al. 2023) through a look-ahead exercise, so as to rapidly achieve the pre-set breeding outcome.

Our study also demonstrated that three backcrosses were sufficient to shift lint yield as high as the RP, while transferring Ren^lon reniform-nematode resistance from a poor agronomic and exotic line into elite genotypic backgrounds. The fourth addition could provide additional gain at the family level, but not in terms of the best-yielding fixed lines (Table S5). For fibre-quality properties, three backcrosses were sufficient to recover the superiority of traits of the RP (Fig. 4). This is consistent with the fact that fibre properties are usually inherited simpler than lint yield in cotton (Liu et al. 2011; Campbell and Myers 2015). The minimal backcross generations required in this study were consistent with the number used for introgression of bacterial blight resistance in cotton (Bayles et al. 2005) and reported for transgenic trait introgression (Zhang 2015; Conaty et al. 2022).

Conclusions

This study demonstrated the effectiveness of two exotic resistance sources to local reniform nematode and identified the superior one, i.e. Ren^barb, for breeding resistant cotton in Australia. Backcrossing integrated with marker-assisted selection for resistance was effective in transferring the resistance into elite genotypic backgrounds, but to regain yield competitiveness of the resultant families, at least three times of repeated backcrossing was required. That, plus further exploitation of within-family variation should ensure to retain and identify lines with the combination of high performance and resistance to reniform nematode. Relying on such an approach, this study successfully stacked resistance together with current transgenic traits and developed resistant transgenic lines with promising performance in both yield and fibre quality. Because they were competitive to the current commercial varieties in non-infested region in our multi-location and season yield testings, we would expect their adoption to provide a cost-effective measure to the infested farms to control and manage reniform nematode and then to ensure higher farm productivity and economic return. This measure should also minimise the future spread and impact of the parasite. We further propose more effective breeding strategies for cotton resistance to reniform nematode, under the vision of further enhanced agronomic performance as well as durable resistance.

Supplementary material

Supplementary material is available online.